The present invention relates to a novel class of compounds, including heteroaryl aminobenzophenone derivatives, which show anti-inflammatory effects, to pharmaceutical compositions containing these compounds, and to their use in the treatment and prophylaxis of inflammatory diseases.
A series of aminobenzophenones (e.g. 4-(2-amino-4-nitrophenylamino)benzophenone) have been described previously (Hussein, F. A. et al., Iraqi J. Sci., 22, 54-66 (1981)). In this publication, however, there is no description of any potential therapeutic use of such compounds. WO 98/32730 discloses aminobenzophenone inhibitors of interleukin 1xcex2 (IL-1xcex2) and tumour necrosis factor xcex1 (TNF-xcex1) secretion in vitro, and indicates the potential utility of these compounds in the treatment of inflammatory diseases in which the production of pro-inflammatory cytokines is involved in the pathogenesis, e.g. asthma, rheumatoid arthritis, psoriasis, contact dermatitis, and atopic dermatitis. Furthermore the A compounds disclosed in WO 98/32730 were tested in vivo for anti-inflammatory properties in the 12-O-tetradecanoylphorbol-13-acetate (TPA) induced murine chronic skin inflammation model (De Young, L. M. et al., Agents Actions 26, 335-341 (1989); Carlson, R. P. et al., Agents Actions 17, 197-204 (1985); Alford, J. G. et al., Agents Action 37, (1992); Stanley, P. L. et al., Skin Pharmacol. 4, 262-271 (1991)). In this chronic skin inflammation model the compounds had the same potency compared to the reference compound hydrocortisone.
It is the object of the present invention is to provide further pharmacologically active benzophenone derivatives which differ structurally from those disclosed in WO 98/32730.
It has surprisingly been found that novel benzophenone derivatives are potent inhibitors of interleukin 1xcex2 (IL-1xcex2) and tumour necrosis factor xcex1 (TNF-xcex1) secretion in vitro, making them potentially useful for the treatment and/or prevention of inflammatory diseases and other conditions in which the secretion and regulation of cytokines or more specifically interleukin 1xcex2 (IL-1xcex2) and tumour necrosis factor xcex1 (TNF-xcex1) are involved in the pathogenesis. The inhibition or downregulation of the cytokines is possibly due to an inhibition of MAP kinases, more specifically the p38 MAP kinase, a stress-activated protein which is an important element in the signal transduction pathway leading to the production of pro-inflammatory cytokines.
Accordingly, the present invention relates to a compound with the general formula I 
wherein R1 is selected from the group consisting of halogen, haloalkyl, hydroxy, hydroxyalkyl, hydroxyalkyloxy, mercapto, cyano, carboxy, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, alkylaryl, alkoxy, aralkoxy, alkylthio, alkoxycarbonyl, alkylcarbonyloxy, alkoxycarbonyloxy, alkylsulfonyloxy, alkyloxysulfonyl, alkylcarbonylamino, aminocarboaminoalkyl, aminosulfonyl, alkylsulfonylamino, alkanoyl, alkylcarbonyl, xe2x80x94NR9R10 or xe2x80x94CONR9R10, wherein R9 and R10 are the same or different and individually represent hydrogen, alkyl or aryl;
R2 represents one or more, same or different substituents selected from the group consisting of hydrogen, halogen, haloalkyl, hydroxy, hydroxyalkyl, hydroxyalkyloxy, mercapto, cyano, carboxy, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, alkylaryl, alkoxy, aralkoxy, alkylthio, alkoxycarbonyl, alkylcarbonyloxy, alkoxycarbonyloxy, alkylsulfonyloxy, alkyloxysulfonyl, alkylcarbonylamino, aminocarboaminoalkyl, aminosulfonyl, alkylsulfonylamino, alkanoyl, alkylcarbonyl, xe2x80x94NR9R10 or xe2x80x94CONR9R10, wherein R9 and R10 are the same or different and individually represent hydrogen, alkyl or aryl;
R3 represents one or more, same or different substituents selected from the group consisting of hydrogen, halogen, haloalkyl, hydroxy, hydroxyalkyl, mercapto, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, alkylaryl, alkoxy, aralkoxy, alkylthio, alkoxycarbonyl, alkylcarbonylamino, alkylcarbonyloxy, alkoxycarbonyloxy, alkylcarbonyl xe2x80x94NR9R10 or xe2x80x94CONR9R10, wherein R9 and R10 are the same or different and individually represent hydrogen, alkyl or aryl;
R4 represents hydrogen, alkyl, alkenyl , alkynyl, cycloalkyl, cycloalkenyl, carboxy or aryl;
R5 represents a heteroaromatic mono- or bicyclic ring system comprising 1-4 heteroatoms, except for triazine, said ring system being optionally substituted by hydrogen, halogen, haloalkyl, hydroxy, hydroxyalkyl, hydroxyalkyloxy, mercapto, cyano, carboxy, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, alkylaryl, alkoxy, aralkoxy, alkylthio, alkoxycarbonyl, alkylcarbonyloxy, alkoxycarbonyloxy, alkylsulfonyloxy, alkyloxysulfonyl, alkylcarbonylamino, aminocarboaminoalkyl, aminosulfonyl, alkylsulfonylamino, alkanoyl, alkylcarbonyl, xe2x80x94NR9R10 or xe2x80x94CONR9R10, wherein R9 and R10 are the same or different and individually represent hydrogen, alkyl or aryl;
X represents oxygen, sulphur, Nxe2x80x94OH or NR11 wherein R11 is hydrogen or alkyl;
or pharmaceutically acceptable salts hydrates, solvates or esters thereof as well as N-oxides wherein an N-atom of R5 is oxidised.
In the present context, the term xe2x80x9calkylxe2x80x9d is intended to indicate a univalent radical derived from straight or branched alkane by removing a hydrogen atom from any carbon atom. The alkyl chain typically comprises 1-10 carbon atoms, in particular 1-6 carbon atoms. The term includes the subclasses normal alkyl (n-alkyl), secondary and tertiary alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec.-butyl, tert.-butyl, pentyl, isopentyl, hexyl and isohexyl.
The term xe2x80x9chaloalkylxe2x80x9d is intended to indicate an alkyl radical as defined above substituted by one or more halogens such as chloro, fluoro, bromo or iodo.
The term xe2x80x9chydroxyalkylxe2x80x9d is intended to indicate an alkyl radical as defined above substituted by one or more hydroxy groups.
The term xe2x80x9calkoxyxe2x80x9d is intended to indicate a radical of formula ORxe2x80x2, wherein Rxe2x80x2 is alkyl as defined above, e.g. methoxy, ethoxy, propoxy, butoxy, etc.
The term xe2x80x9chydroxyalkyloxyxe2x80x9d is intended to indicate an alkoxy group as defined above substituted by one or more hydroxy groups.
The term xe2x80x9calkenylxe2x80x9d is intended to indicate a mono-, di-, tri-, tetra- or pentaunsaturated alkane radical typically comprising 2-10 carbon atoms, in particular 2-6 carbon atoms, e.g. ethenyl, propenyl, butenyl, pentenyl or hexenyl. The term xe2x80x9calkynylxe2x80x9d is intended to indicate an alkane radical comprising 1-5 triple Cxe2x80x94C bonds, the alkane chain typically comprising 2-10 carbon atoms, in particular 2-6 carbon atoms, such as ethynyl, propynyl, butynyl, pentynyl or hexynyl.
The term xe2x80x9calkoxycarbonylxe2x80x9d is intended to indicate a radical of formula xe2x80x94COORxe2x80x2 wherein Rxe2x80x2 is alkyl as defined above, e.g. methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, etc.
The term xe2x80x9ccycloalkylxe2x80x9d is intended to indicate a saturated cycloalkane radical typically comprising 3-10 carbon atoms, in particular 3-8 carbon atoms, e.g. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. The term xe2x80x9ccycloalkenylxe2x80x9d is intended to indicate mono-, di- tri- or tetraunsaturated cycloalkane radicals, e.g. cyclopropenyl, cyclobutenyl, cyclopentenyl or cyclohexenyl. The term xe2x80x9cheterocycloalkylxe2x80x9d is intended to indicate a cycloalkane radical as defined above, comprising one or more heteroatoms selected from O, N, or S.
The term xe2x80x9carylxe2x80x9d is intended to include radicals of carbocyclic aromatic rings, in particular 5- or 6-membered rings, optionally fused bicyclic rings, e.g. phenyl or naphthyl. The term xe2x80x9cheteroarylxe2x80x9d is intended to include radicals of heterocyclic aromatic rings, in particular 5- or 6-membered rings with 1-4 heteroatoms selected from O, S and N, or optionally fused bicyclic rings with 1-4 heteroatoms, e.g. pyridyl, quinolyl, isoquinolyl, indolyl, tetrazolyl, thiazolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thienyl, pyrazinyl, isothiazolyl, benzimidazolyl and benzofuranyl.
The term xe2x80x9calkylcarbonyloxyxe2x80x9d refers to a radical of formula Rxe2x80x2xe2x80x94COOxe2x80x94, wherein Rxe2x80x2 is alkyl as indicated above. The term xe2x80x9calkoxycarbonyloxyxe2x80x9d refers to a radical of formula Rxe2x80x2Oxe2x80x94COOxe2x80x94, wherein Rxe2x80x2 is alkyl as defined above. The term xe2x80x9calkylsulfonyloxyxe2x80x9d refers to a radical of formula Rxe2x80x2xe2x80x94(SO2)xe2x80x94Oxe2x80x94, wherein Rxe2x80x2 is alkyl as defined above. The term xe2x80x9calkyloxysulfonylxe2x80x9d refers to a radical of formula Rxe2x80x2Oxe2x80x94(SO2)xe2x80x94, wherein Rxe2x80x2 is alkyl as defined above.
The term xe2x80x9caralkylxe2x80x9d is intended to indicate an aromatic ring with an alkyl side chain as defined above, e.g. benzyl. The term xe2x80x9calkylarylxe2x80x9d is intended to indicate an alkyl radical as defined above comprising an aromatic side chain.
The term xe2x80x9chalogenxe2x80x9d is intended to indicate fluoro, chloro, bromo or iodo. The term xe2x80x9calkylthioxe2x80x9d is intended to indicate a radical of the formula xe2x80x94SR, where R is alkyl as defined above and includes methylthio, ethylthio, n-propylthio, and 2-propylthio.
The term xe2x80x9calkylaminoxe2x80x9d is intended to indicate a radical of the formula xe2x80x94NHR or xe2x80x94NR2, where R is alkyl as defined above having from 1-6 carbon atoms and includes, for example, methylamino, dimethylamino, di-(n-propyl)amino, and n-butyl(ethyl)amino.
The term xe2x80x9ccarbamoylxe2x80x9d is intended to indicate the group xe2x80x94OCONH2, xe2x80x94OCONHR, and xe2x80x94OCONRRxe2x80x2 where R and Rxe2x80x2 represent alkyl as defined above.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d is intended to indicate alkali metal or alkaline earth metal salts, for instance sodium, potassium, magnesium or calcium salts, as well as silver salts and salts with suitable organic or inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric, nitric, phosphoric, acetic, lactic, maleic, phthalic, citric, propionic, benzoic, glutaric, gluconic, methanesulfonic, salicylic, succinic, tartaric, toluenesulfonic, sulfamic or fumaric acid.
The term xe2x80x9cpharmaceutically acceptable estersxe2x80x9d is intended to indicate easily hydrolysable esters such as alkanoyloxyalkyl, aralkanoyloxyalkyl, aroyloxyalkyl, e.g. acetoxymethyl, pivaloyloxymethyl, benzoyloxymethyl esters and the corresponding 1xe2x80x2-oxyethyl derivatives, or alkoxycarbonyloxyalkyl esters, e.g. methoxycarbonyloxymethyl esters and ethoxycarbonyloxymethyl esters and the corresponding 1xe2x80x2-oxyethyl derivatives, or lactonyl esters, e.g. phthalidyl esters, or dialkylaminoalkyl esters, e.g. dimethylaminoethyl esters. Easily hydrolysable esters include in vivo hydrolysable esters of the compounds of formula I. Such esters may be prepared by conventional methods known to persons skilled in the art, such as method disclosed in GB patent No. 1 490 852 incorporated herein by reference.
xe2x80x9cp38 MAP kinasexe2x80x9d is a stress-activated protein kinase existing in several isoforms (p38xcex1, p38xcex2, p38xcex22, p38xcex3 and p38xcex4). The p38 MAP kinase is activated by different stimuli including heat, chemical, osmotic, pH and oxidative stress, growth factor withdrawal, high or low glucose and ultraviolet radiation. p38 is also stimulated by agents that mediate the initial physiological response to injury, infection and inflammation, such as LPS and pro-inflammatory cytokines IL-1xcex2, TNF-xcex1, FasL, CD40L and TGF-xcex2. Like other MAP kinases, p38 is phosphorylated by kinases, including MKK3, MEK6 and MKK6, on a threonine and tyrosine in an activation loop (Thr-Xaa-Tyr) close to the ATP and substrate binding site. In turn, p38 phosphorylates and activates the serine-threonine protein kinases MAPKAP kinase-2, MAPKAP kinase-3, MAPKAP kinase-5, MNK-1 and MSK-1. It has been established that activation of p38 regulates cytokine biosynthesis in many cell types either directly by phosphorylating and activating transcription factors involved in the expression of cytokines or indirectly, e.g. by phosphorylating MSK-1 which, when activated, activates the transcription factor CREB. It has also been shown that certain pyridinyl imidazoles, e.g. SB203580, inhibit the production of IL-1xcex2 and TNF-xcex1 from LPS-treated human monocytes, are inhibitors of p38 kinase. It has therefore been concluded that p38 constitutes a potentially highly interesting target for the development of anti-inflammatory compounds (cf. J C Lee et al., Immunopharmacology 47, 2000, pp. 185-201 and references reviewed 1t therein; P R Young, xe2x80x9cSpecific Inhibitors of p38 MAP kinasexe2x80x9d in Signaling Networks and Cell Cycle Control: The Molecular Basis of Cancer and Other Diseases, J S Gutkind (Ed.), Humana Press, Inc., Totowa, N.J., and references reviewed therein).
In compounds of formula I,
R1 preferably represents a substituent selected from the group consisting of halogen, hydroxy, mercapto, trifluoromethyl, amino, (C1-C6)alkyl, (C2-C6)alkenyl, (C1-C6)alkoxy, (C1-C6)alkylthio, (C1-C6)alkylamino, (C1-C6) alkoxycarbonyl, cyano, xe2x80x94CONH2, phenyl, and nitro, in particular fluoro, chloro, bromo, hydroxy, trifluoromethyl, amino, (C1-C3)alkyl, (C2-C3)alkenyl, (C1-C3)alkoxy, (C1-C3) alkoxycarbonyl, cyano, or xe2x80x94CONH2;
R2 represents one or more, same or different substituents selected from the group consisting of hydrogen, halogen, hydroxy, mercapto, trifluoromethyl, amino, (C1-C6)alkyl, (C2-C6)alkenyl, (C1-C6)alkoxy, (C1-C6)alkylthio, (C1-C6)alkylamino, (C1-C6)alkoxycarbonyl, cyano, xe2x80x94CONH2, phenyl, and nitro; in particular hydrogen, fluoro, chloro, bromo, hydroxy, trifluoromethyl, amino, (C1-C3)alkyl, (C2-C3)aikenyl, (C1-C3)alkoxy, (C1-C3) alkoxycarbonyl, cyano, or xe2x80x94CONH2;
R3 represents one or more, same or different substituents selected from the group consisting of hydrogen, halogen, hydroxy, mercapto, trifluoromethyl, amino, (C1-C6)alkyl, (C2-C6)alkenyl, (C1-C6)alkoxy, (C1-C6)alkylthio, (C1-C6)alkylamino, (C1-C6) alkoxycarbonyl, cyano, xe2x80x94CONH2, phenyl, and nitro, in particular hydrogen, fluoro, chloro, bromo, hydroxy, trifluoromethyl, amino, (C1-C3)alkyl, (C2-C3)alkenyl, and (C1-C3)alkoxy;
R4 represents hydrogen, (C1-C6)alkyl, (C2-C6) alkenyl, or (C3-C6) cycloalkyl or cycloalkenyl, in particular hydrogen, (C1-C4) alkyl, or (C2-C4) alkenyl;
X represents oxygen or NH.
In the mono- or bicyclic heteroaromatic ring system represented by R5, the heteroatom(s) may be selected from N, S or O. Each ring preferably comprises 5 or 6 ring atoms. Examples of suitable heteroaromatic ring systems are selected from the group consisting of pyridyl, pyrazinyl, pyrimidinyl, quinolyl, isoquinolyl, quinazolinyl, pyridazinyl, phthalazinyl, purinyl, quinoxalyl, allopurinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzoxazolyl, benzothienyl, isobenzothienyl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl, indolizinyl, isoindolyl, indolyl, indazolyl, triazolyl, oxazolyl, thiadiazolyl, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl or isoxazolyl.
R5 is preferably selected from heteroaromatic ring systems comprising 1 or 2 nitrogen atoms, e.g. rings of the structures: 
The R5 ring-systems may optionally be oxidised to the corresponding N-oxides as exemplified below for the isoquinoline ring system: 
R6 and R7 represent one or more, same or different substituents selected from the group consisting of hydrogen, halogen, hydroxy, mercapto, trifluoromethyl, cyano, carboxy, carbamoyl, amino, nitro, (C1-C10)alkyl, (C2-C10)alkenyl, (C3-C8)cycloalkyl or -cycloalkenyl, (C1-C10)alkoxy, (C1-C10)alkylthio, (C1-C10) alkoxycarbonyl, and phenyl. In particular R6 and R7 represent one or more, same or different substituents selected from the group consisting of hydrogen, fluoro, chloro, bromo, hydroxy, trifluoromethyl, amino, (C1-C6)alkyl, (C2-C6)alkenyl, (C1-C6)alkoxy, (C1-C6) alkoxycarbonyl, cyano, carboxy, and xe2x80x94CONH2; and 
R8 represents hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, or (C3-C6)cycloalkyl or -cycloalkenyl.
In compounds of formula I it is more preferred that
R1 represents a substituent selected from the group consisting of fluoro, chloro, bromo, hydroxy, methyl, and methoxy;
R2 represents one or more, same or different substituents selected from the group consisting of hydrogen, fluoro, chloro, bromo, hydroxy, trifluoromethyl, methyl, ethyl, and methoxy;
R3 represents one or more, same or different substituents selected from the group consisting of hydrogen, fluoro, chloro, bromo, hydroxy, methyl, and methoxy;
R4 represents hydrogen, methyl, or ethyl;
R5 is selected from the group consisting of substituted or non-substituted 3-pyridyl, 2-pyridyl, 3-quinolyl, 4-isoquinolyl, 4-indolyl, 5-indolyl, 6-indolyl and 7-indolyl moieties, and the corresponding N-oxides;
R6 and R7 represent one or more, same or different substituents selected from the group consisting of hydrogen, fluoro, chloro, bromo, hydroxy, methyl, methoxy, cyano, and carboxy;
R8 represents hydrogen, (C1-C4)alkyl, (C2-C6)alkenyl, such as allyl; and more preferably, R8 represents hydrogen, methyl, ethyl, allyl, propyl, or t-butyl; and/or
X represents oxygen.
The phenyl group of R1, R2, R3, R6 and R7 may optionally be substituted, e.g. with hydroxy, amino, nitro, cyano or halogen, preferably fluoro or chloro.
Specific compounds of the invention are:
2-Chloro-2xe2x80x2-methyl-4-(4-pyridylamino)benzophenone (Compound 101), 2-Chloro-2xe2x80x2-methyl-4-(2-pyridylamino)benzophenone (Compound 102), 2-Chloro-2xe2x80x2-methyl-4-(5-nitro-2-pyridylamino)benzophenone (Compound 103), 4-(6-Amino-5-nitro-2-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone (Compound 104), 6-Chloro-2-(3-chloro-4-(2-methylbenzoyl)phenylamino)isonicotinic acid (Compound 105), 4-(6-carbonitrile-2-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone (Compound 106), 2-Chloro-2xe2x80x2-methyl-4-(3-pyridylamino)benzophenone (Compound 107), 2-Chloro-4-(5,6-diamino-2-pyridylamino)-2xe2x80x2-methylbenzophenone (Compound 108), 2-Chloro-2xe2x80x2-methyl-4-(3-nitro-2-pyridylamino)benzophenone (Compound 109), 4-(3-Amino-2-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone (Compound 110), 4-(5-Amino-2-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone (Compound 111), 2-Chloro-4-(4-isoquinolylamino)-2xe2x80x2-methylbenzophenone (Compound 114), t-Butyl 5-(3-chloro-4-(2-methylbenzoyl)phenylamino)nicotinoate (Compound 115, ) 2-Chloro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)benzophenone (Compound 116), 2-Chloro-2xe2x80x2-methyl-4-(6-methyl-3-pyridylamino)benzophenone (Compound 117), 4-(5-Bromo-3-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone (Compound 118), 4-(5-Carbonitrile-3-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone (Compound 119), 4-(3-Bromo-2-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone (Compound 120), 2-Chloro-2xe2x80x2-methyl-4-(8-quinolylamino)benzophenone (Compound 121), 2-Chloro-4-(6-ethoxy-3-pyridylamino)-2xe2x80x2-methylbenzophenone (Compound 122), 4- (4-Bromo-1-isoquinolylamino)-2-chloro-2xe2x80x2-methylbenzophenone (Compound 123), 2-Chloro-2xe2x80x2-methyl-4-(2- methyl-5-trifluoromethyl-3-pyridylamino)benzophenone (Compound 124), 2-Chloro-2xe2x80x2-methyl-4-(3-quinolylamino)benzophenone (Compound 125), 2-Chloro-4-(4-ethoxy-3-pyridylamino)-2xe2x80x2-methylbenzophenone (Compound 126), 2-Chloro-4-(2-ethoxy-3-pyridylamino)-2xe2x80x2-methylbenzophenone (Compound 127), 2-Chloro-2xe2x80x2-methyl-4-(2-methyl-6-quinolylamino)benzophenone (Compound 129), 2-Chloro-4-(7-chloro-4-quinolylamino)-2xe2x80x2-methylbenzophenone (Compound 130), 2-Chloro-2xe2x80x2-methyl-4-(2-quinolylamino)benzophenone (Compound 131), 2-Chloro-2xe2x80x2-methyl-4-(4-quinolylamino) benzophenone(Compound 132), 2-Chloro-2xe2x80x2-methyl-4-(2-methyl-7-indolylamino)benzophenone (Compound 133), 2-Chloro-2xe2x80x2-methyl-4-(4-methyl-5-indolylamino)benzophenone (Compound 134), 2-Chloro-2xe2x80x2,5xe2x80x2-dimethyl-4-(4-methyl-3-pyridylamino)benzophenone (Compound 135), 2-Chloro-2xe2x80x2,5xe2x80x2-dimethyl-4-(4-isoquinolylamino)benzophenone (Compound 136), 2-Chloro-4-(4-isoquinolylamino)-2xe2x80x2,4xe2x80x2,5xe2x80x2-trimethylbenzophenone (Compound 137), 2,3xe2x80x2-Dichloro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)benzophenone (Compound 138), 2-Fluoro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)benzophenone (Compound 139), 2,4xe2x80x2-Dichloro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)benzophenone (Compound 140), 2-Chloro-4xe2x80x2-fluoro-4-(4-isoquinolylamino)-2xe2x80x2-methylbenzophenone (Compound 141), 4xe2x80x2-n-Butyl-2-chmoro-4-(4-isoquinolyl amino)-2xe2x80x2-methylbenzophenone (Compound 142), 2-Chloro-4-(5-isoquinolylamino)-2xe2x80x2-methylbenzophenone (Compound 143), 2-Chloro-4-(4-isoquinolylamino)-4xe2x80x2-methoxy-2xe2x80x2-methylbenzophenone (Compound 144), 2-Fluoro-4-(4-isoquinolylamino)-4xe2x80x2-methoxy-2xe2x80x2-methylbenzophenone (Compound 145), 2,4xe2x80x2-Dichloro-2xe2x80x2-methyl-4-(1-methyl-7-indolylamino)benzophenone (Compound 146), 2-Chloro-4-(1-methyl-7-indolylamino)-2xe2x80x2,4xe2x80x2,5xe2x80x2-trimethylbenzophenone (Compound 147), 2-Chloro-2xe2x80x2,5xe2x80x2-dimethyl-4-(1-methyl-7-indolylamino)benzophenone (Compound 148), 2-Chloro-4-(3-ethoxy-4-isoquinolylamino)-2xe2x80x2-methylbenzophenone (Compound 149), 2-Chloro-4-(1-ethoxy-4-isoquinolylamino)-2xe2x80x2-methylbenzophenone (Compound 150), 4-(2-Benzoxazolylamino)-2-chloro-2xe2x80x2-methylbenzophenone (Compound 151), 4-(1-Methyl-2-benzimidazolylamino)-2-chloro-2xe2x80x2-methylbenzophenone (Compound 152), 4-(2-Benzothiazolylamino)-2-chloro-2xe2x80x2-methylbenzophenone (Compound 153), 2-Chloro-2xe2x80x2-methyl-4-(2-pyrimidylamino)benzophenone (Compound 154), 2-Chloro-2xe2x80x2-methyl-4-(7-methyl-purin-6-ylamino)benzophenone (Compound 155), 2-Chloro-2xe2x80x2-methyl-4-(2-methyl-5-benzothiazolylamino)benzophenone (Compound 156), 2-Chloro-2xe2x80x2-methyl-4-(pyrazin-2-ylamino)benzophenone (Compound 157), 2-Chloro-2xe2x80x2-methyl-4-(5-pyrimidylamino)benzophenone (Compound 158), 2-Chloro-2xe2x80x2-methyl-4-(5-nitro-2-thiazolylamino)benzophenone (Compound 159), 2-Chloro-2xe2x80x2-methyl-4-((4-methyl-3-nitro-(1,2,4-triazol-5-ylamino))benzophenone (Compound 160)
and the N-oxides thereof wherein the nitrogen atom of the heterocyclic R5 substituent is specifically oxidised.
More preferred compounds are:
2-Chloro-2xe2x80x2-methyl-4-(3-pyridylamino)benzophenone (Compound 107), 4-(3-Amino-2-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone (Compound 110), 2-Chloro-4-(4-isoquinolylamino)-2xe2x80x2-methylbenzophenone (Compound 114), 2-Chloro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)benzophenone (Compound 116), 2-Chloro-2xe2x80x2-methyl-4-(6-methyl-3-pyridylamino)benzophenone (Compound 117), 4-(5-Bromo-3-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone (Compound 118), 2-Chloro-2xe2x80x2-methyl-4-(8-quinolylamino)benzophenone (Compound 121), 2-Chloro-4-(6-ethoxy-3-pyridylamino)-2xe2x80x2-methylbenzophenone (Compound 122), 2-Chloro-2xe2x80x2-methyl-4-(3-quinolylamino)benzophenone (Compound 125), 2-Chloro-4-(4-ethoxy-3-pyridylamino)-2xe2x80x2-methylbenzophenone (Compound 126), 2-Chloro-2xe2x80x2-methyl-4-(1-methyl-7-indolylamino)benzophenone (Compound 133), 2-Chloro-2xe2x80x2-methyl-4-((1-methyl-5-indolylamino)benzophenone (Compound 134), 2-Chloro-2xe2x80x2,5xe2x80x2-dimethyl-4-(4-methyl-3-pyridylamino)benzophenone (Compound 135) 2-Chloro-2xe2x80x2,5xe2x80x2-dimethyl-4-(4-isoquinolylamino)benzophenone (Compound 136), 2-Chloro-4-(4-isoquinolylamino)-2xe2x80x2,4xe2x80x2,5xe2x80x2-trimethylbenzophenone (Compound 137), 2-Fluoro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)benzophenone (Compound 139), 2,4xe2x80x2-Dichloro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)benzophenone (Compound 140), 2-Chloro-4xe2x80x2-fluoro-4-(4-isoquinolylamino)-2xe2x80x2-methylbenzophenone (Compound 141), 2-Chloro-4-(5-isoquinolylamino)-2xe2x80x2-methylbenzophenone (Compound 143), 2-Chloro-4-(4-isoquinolylamino)-4xe2x80x2-methoxy-2xe2x80x2-methylbenzophenone (Compound 144), 2-Fluoro-4-(4-isoquinolylamino)-4xe2x80x2-methoxy-2xe2x80x2-methylbenzophenone (Compound 145), 2-Chloro-4-((3-ethoxy)-4-isoquinolylamino)-2xe2x80x2-methylbenzophenone (Compound 149), 2-Chloro-4-((1-ethoxy)-4-isoquinolylamino)-2xe2x80x2-methylbenzophenone (Compound 150)
and the N-oxides
2-Chloro-2xe2x80x2-methyl-4((2-pyridyl-N-oxide)amino)benzophenone (Compound 112), 2-Chloro-2xe2x80x2-methyl-4((3-pyridyl-N-oxide)amino)benzophenone (Compound 113), 2-Chloro-4((4-isoquinolyl-N-oxide)amino)-2xe2x80x2-methylbenzophenone (Compound 128),
Compounds of formula Ia wherein X=S and wherein R1, R2, R3, R4, R5, R6, R7 and R8 are as indicated above, and compounds of formula Ib wherein Xxe2x95x90Nxe2x80x94OH and wherein R1, R2 R3, R4, R5, R6, R7 and R8 are as indicated above are also generally preferred. 
Specific compounds of formula Ia are:
2-Chloro-2xe2x80x2-methyl-4-(4-pyridylamino)thiobenzophenone, 2-Chloro-2xe2x80x2-methyl-4-(2-pyridylamino)thiobenzophenone, 2-Chloro-2xe2x80x2-methyl-4-(5-nitro-2-pyridylamino)thiobenzophenone, 4-(6-Amino-5-nitro-2-pyridylamino)-2-chloro-2xe2x80x2-methyl(thiobenzophenone), 6-Chloro-2-(3-chloro-4-(2-methylthiobenzoyl)phenylamino)isonicotinic acid, 4-(6-carbonitrile-2-pyridylamino)-2-chloro-2xe2x80x2-methyl(thiobenzophenone), 2-Chloro-2xe2x80x2-methyl-4-(3-pyridylamino)thiobenzophenone, 2-Chloro-4-(5,6-diamino-2-pyridylamino)-2xe2x80x2-methyl(thiobenzophenone), 2-Chloro-2xe2x80x2-methyl-4-(3-nitro-2-pyridylamino)thiobenzophenone, 4-(3-Amino-2-pyridylamino)-2-chloro-2xe2x80x2-methyl(thiobenzophenone), 4-(5-Amino-2-pyridylamino)-2-chloro-2xe2x80x2-methyl(thiobenzophenone), 2-Chloro-4-(4-isoquinolylamino)-2xe2x80x2-methyl(thiobenzophenone), t-Butyl 5-(3-chloro-4-(2-methylthiobenzoyl)phenylamino)nicotinoate, 2-Chloro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)thiobenzophenone, 2-Chloro-2xe2x80x2-methyl-4-(6-methyl-3-pyridylamino)thiobenzophenone, 4-(5-Bromo-3-pyridylamino)-2-chloro-2xe2x80x2-methyl(thiobenzophenone), 4-(5-Carbonitrile-3-pyridylamino)-2-chloro-2xe2x80x2-methyl(thiobenzophenone), 4-(3-Bromo-2-pyridylamino)-2-chloro-2xe2x80x2-methyl(thiobenzophenone), 2-Chloro-2xe2x80x2-methyl-4-(8-quinolylamino)thiobenzophenone, 2-Chloro-4-(6-ethoxy-3-pyridylamino)-2xe2x80x2-methyl(thiobenzophenone), 4-(4-Bromo-1-isoquinolylamino)-2-chloro-2xe2x80x2-methyl(thiobenzophenone), 2-Chloro-2xe2x80x2-methyl-4-(2-methyl-5-trifluoromethyl-3-pyridylamio)thiobenzophenone, 2-Chloro-2xe2x80x2-methyl-4-(3-quinolylamino)thiobenzophenone, 2-Chloro-4-(4-ethoxy-3-pyridylamino)-2xe2x80x2-methyl(thiobenzophenone), 2-Chloro-4-(2-ethoxy-3-pyridylamino)-2xe2x80x2-methyl(thiobenzophenone), 2-Chloro-2xe2x80x2-methyl-4-(2-methyl-6-quinolylamino)thiobenzophenone, 2-Chloro-2xe2x80x2-methyl-4-(2-quinolylamino)thiobenzophenone, 2-Chloro-4-(7-chloro-4-quinolylamino)-2xe2x80x2-methyl(thiobenzophenone), 2-Chloro-2xe2x80x2-methyl-4-(4-quinolylamino)thiobenzophenone, 2-Chloro-2xe2x80x2-methyl-4-(1-methyl-7-indolylamino)thiobenzophenone, and 2-Chloro-2xe2x80x2-methyl-4-(1-methyl-5-indolylamino)thiobenzophenone, 2-Chloro-2xe2x80x2,5xe2x80x2-dimethyl-4-(4-methyl-3-pyridylamino)thiobenzophenone, 2-Chloro-2xe2x80x2,5xe2x80x2-dimethyl-4-(4-isoquinolylamino)thiobenzophenone, 2-Chloro-4-(4-isoquinolylamino)-2xe2x80x2,4xe2x80x2,5xe2x80x2-trimethyl(thiobenzophenone), 2,3xe2x80x2-Dichloro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)thiobenzophenone, 2-Fluoro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)thiobenzophenone, 2,4xe2x80x2-Dichloro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)thiobenzophenone, 2-Chloro-4xe2x80x2-fluoro-4-(4-isoquinolylamino)-2xe2x80x2-methyl(thiobenzophenone), 4xe2x80x2-n-Butyl-2-chloro-4-(4-isoquinolylamino)-2xe2x80x2-methyl(thiobenzophenone), 2-Chloro-4-(5-isoquinolylamino)-2xe2x80x2-methyl(thiobenzophenone), 2-Chloro-4-(4-isoquinolylamino)-4xe2x80x2-methoxy-2xe2x80x2-methyl(thiobenzophenone), 2-Fluoro-4-(4-isoquinolylamino)-4xe2x80x2-methoxy-2xe2x80x2-methyl(thiobenzophenone), 2,4xe2x80x2-Dichloro-2xe2x80x2-methyl-4-(1-methyl-7-indolylamino)thiobenzophenone, 2-Chloro-4-(1-methyl-7-indolylamino)-2xe2x80x2,4xe2x80x2,5xe2x80x2-trimethyl(thiobenzophenone), 2-Chloro-2xe2x80x2,5xe2x80x2-dimethyl-4-(1-methyl-7-indolylamino)thiobenzophenone, 2-Chloro-4-(3-ethoxy-4-isoquinolylamino)-2xe2x80x2-methyl(thiobenzophenone), 2-Chloro-4-(1-ethoxy-4-isoquinolylamino)-2xe2x80x2-methyl(thiobenzophenone), 4-(2-Benzoxazolylamino)-2-chloro-2xe2x80x2-methyl(thiobenzophenone), 4-(1-Methyl-2-benzimidazolylamino)-2-chloro-2xe2x80x2-methyl(thiobenzophenone), 4-(2-Benzothiazolylamino)-2-chloro-2xe2x80x2-methyl(thiobenzophenone), 2-Chloro-2xe2x80x2-methyl-4-(2-pyrimidylamino)thiobenzophenone, 2-Chloro-2xe2x80x2-methyl-4-(2-methyl-purin-6-ylamino)thiobenzophenone, 2-Chloro-2xe2x80x2-methyl-4-(2-methyl-5-benzothiazolylamino)thiobenzophenone, 2-Chloro-2xe2x80x2-methyl-4-(pyrazin-2-ylamino)thiobenzophenone, 2-Chloro-2xe2x80x2-methyl-4-(5-pyrimidylamino)thiobenzophenone
and salts thereof with pharmaceutically acceptable acids, hydrates and solvates.
Specific compound of formula Ib are:
2-Chloro-2xe2x80x2-methyl-4-(4-pyridylamino)benzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(2-pyridylamino)benzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(5-nitro-2-pyridylamino)benzophenone oxime, 4-(6-Amino-5-nitro-2-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone oxime, 6-Chloro-2-((3-chloro-4-((hydroxyimino)(2-methylphenyl)methyl))phenylamino)isonicotinic acid, 4-(6-carbonitrile-2-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(3-pyridylamino)benzophenone oxime, 2-Chloro-4-(5,6-diamino-2-pyridylamino)-2xe2x80x2-methylbenzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(3-nitro-2-pyridylamino)benzophenone oxime, 4-(3-Amino-2-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone oxime, 4-(5-Amino-2-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone oxime, 2-Chloro-4-(4-isoquinolylamino)-2xe2x80x2-methylbenzophenone oxime, t-Butyl 5-((3-chloro-4-((hydroxyimino) (2-methylphenyl)methyl))phenylamino)nicotinoate, 2-Chloro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)benzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(6-methyl-3-pyridylamino)benzophenone oxime, 4-(5-Bromo-3-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone oxime, 4-(5-Carbonitrile-3-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone oxime, 4-(3-Bromo-2-pyridylamino)-2-chloro-2xe2x80x2-methylbenzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(8-quinolylamino)benzophenone oxime, 2-Chloro-4-(6-ethoxy-3-pyridylamino)-2xe2x80x2-methylbenzophenone oxime, 4-(4-Bromo-1-isoquinolylamino)-2-chloro-2xe2x80x2-methylbenzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(2-methyl-5-trifluoromethyl-3-pyridylamino)benzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(3-quinolylamino)benzophenone oxime, 2-Chloro-4-(4-ethoxy-3-pyridylamino)-2xe2x80x2-methylbenzophenone oxime, 2-Chloro-4-(2-ethoxy-3-pyridylamino)-2xe2x80x2-methylbenzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(2-methyl-6-quinolylamino)benzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(2-quinolylamino)benzophenone oxime, 2-Chloro-4-(7-chloro-4-quinolylamino)-2xe2x80x2-methylbenzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(4-quinolylamino)benzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(1-methyl-7-indolylamino)benzophenone oxime, and 2-Chloro-2xe2x80x2-methyl-4-(1-methyl-5-indolylamino)benzophenone oxime, 2-Chloro-2xe2x80x2,5xe2x80x2-dimethyl-4-(4-methyl-3-pyridylamino)benzophenone oxime, 2-Chloro-2xe2x80x2,5xe2x80x2-dimethyl-4-(4-isoquinolylamino)benzophenone oxime, 2-Chloro-4-(4-isoquinolylamino)-2xe2x80x2,4xe2x80x2,5xe2x80x2-trimethylbenzophenone oxime, 2,3xe2x80x2-Dichloro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)benzophenone oxime, 2-Fluoro-2xe2x80x2-methyl-4-(4-methyl-3-pyridylamino)benzophenone oxime, 2,4xe2x80x2-Dichloro-2xe2x80x2-methyl-4-(4- methyl-3-pyridylamino) benzophenone oxime, 2-Chloro-4xe2x80x2-fluoro-4-(4-isoquinolylamino)-2xe2x80x2-methylbenzophenone oxime, 4xe2x80x2-n-Butyl-2-chloro-4-(4-isoquinolylamino)-2xe2x80x2-methylbenzophenone oxime, 2-Chloro-4-(5-isoquinolylamino)-2xe2x80x2-methylbenzophenone oxime, 2-Chloro-4-(4-isoquinolylamino)-4xe2x80x2-methoxy-2xe2x80x2-methylbenzophenone oxime, 2-Fluoro-4-(4-isoquinolylamino)-4xe2x80x2-methoxy-2xe2x80x2-methylbenzophenone oxime, 2,4xe2x80x2-Dichloro-2xe2x80x2-methyl-4-(1-methyl-7-indolylamino)benzophenone oxime, 2-Chloro-4-(1-methyl-7-indolylamino)-2xe2x80x2,4xe2x80x2,5xe2x80x2-trimethylbenzophenone oxime, 2-Chloro-2xe2x80x2,5xe2x80x2-dimethyl-4-(1-methyl-7-indolylamino)benzophenone oxime, 2-Chloro-4-(3-ethoxy-4-isoquinolylamino)-2xe2x80x2-methylbenzophenone oxime, 2-Chloro-4-(1-ethoxy-4-isoquinolylamino)-2xe2x80x2-methylbenzophenone oxime, 4-(2-Benzoxazolylamino)-2-chloro-2xe2x80x2-methylbenzophenone oxime, 4-(1-Methyl-2-benzimidazolylamino)-2-chloro-2xe2x80x2-methylbenzophenone oxime, 4-(-2-Benzothiazolylamino)-2-chloro-2xe2x80x2-methylbenzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(2-pyrimidylamino)benzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(7-methyl-purin-6-ylamino)benzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(2-methyl-5-benzothiazolylamino)benzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(pyrazin-2-ylamino)benzophenone oxime, 2-Chloro-2xe2x80x2-methyl-4-(5-pyrimidylamino)benzophenone oxime,
and salts thereof with pharmaceutically acceptable acids, hydrates and solvates
Compounds of general formula Ic 
wherein R1, R2, R4, R5, R6, R7, R8 and X are as indicated above, and R3 represents (C1-C3)alkyl, fluoro, chloro, bromo, methoxy, and hydroxy, and salts thereof with pharmaceutically acceptable acids, hydrates and solvates, are generally preferred.
In formula Ic, R1 preferably represents methyl or halogen, more preferably F or Cl; R2 represents one or more substituents, preferably hydrogen, halogen, (C1-C3)alkyl, methoxy or ethoxy, and R3 represents methyl, methoxy or chloro.
To study the effect of the compound of the present invention in vitro the inhibition of the IL-1xcex2 and TNF-xcex1 secretion was measured using the following procedure:
Cytokine production was measured in the media from lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells. The mononuclear cells were isolated from human peripheral blood by Lymphoprep(copyright) (Nycomed, Norway) fractionation and suspended in RPMI 1640 (growth medium) with foetal calf serum (FCS, 2%), at a concentration of 5xc3x97105 cells/ml. The cells were incubated in 24-well tissue culture plates in 1 mL aliquots. Test compounds were dissolved in dimethylsulfoxide (DMSO, 10 mM) and were diluted with the medium. Compounds were added to the cells for 30 minutes, then LPS (1 mg/mL final concentration) was added. The plates were incubated for 18 hours, and the concentration of IL-1xcex2 and TNF-xcex1 in the medium was determined by enzyme-linked immunosorbent assays. The median inhibitory concentrations (IC50) of the compounds were calculated. The results are shown in Table 1.
The compounds of the present invention also show similar activities in the ability to inhibit PMN (polymorphonuclear) superoxide secretion which is also indicative of potentially useful anti-inflammatory drugs. The compounds were tested using the following procedure:
Human polymorphonuclear (PMN) granulocytes were isolated from human blood by dextran sedimentation, Lymphoprep(copyright) fractionation and hypotonic lysis of contaminating erythrocytes.
Superoxide anion generation was measured as the superoxide dismutase inhibitable reduction of ferricytochrome C (Madhu, S. B. et al, Inflammation, 16, 241, (1992)). The cells were suspended in Hanks"" balanced salt solution, and incubated for 10 minutes at 37xc2x0 C. with test compounds. The cells were primed by the addition of TNF-xcex1 (3 ng/mL final concentration) for 10 minutes, and then ferricytochrome C, (final concentration 750 xcexcg/mL), bovine serum albumin (BSA, final concentration 1 mg/mL) and formylmethionyl-leucyl-phenylalanine (fMLP, final concentration 10xe2x88x927 M) were added for 3 minutes. The cells were chilled on ice, and were spun down. The optical densities in the cell-free supernatant was measured in a spectrophotometer. The median inhibitory concentration (IC50) of the compounds was calculated. The results, together with log P of the testet compounds are shown in Table 1.
Ref. comp.: (4-(2-aminophenylamino)-2-chloro-2xe2x80x2-methylbenzophenone, Compound 156 disclosed in WO 98/32730.
These results show that the compounds of the present invention are able to inhibit the production of IL-1xcex2, TNF-xcex1 and PMN-superoxide, and showing a pharmacological activity comparable to a reference compound, thus making them potentially useful in the treatment of inflammatory diseases. The lower log P values of the compounds of the present invention reflects a lower lipophilicity, which indicate compounds with an improved bioavailability as compared to the reference compound.
Cell Culture
COS-1 cells (derived from African green monkey kidney fibroblast-like cell containing wild-type T antigen under control of the SV40 promotor) were obtained from ATCC (ATCC no. CRL-1650) and grown in growth medium (DMEM without phenolred, 10% FCS, 2 mM L-glutamine, 100U penicillin and 100 xcexcg streptomycin/ml) at 37xc2x0 C. with 5% CO2. The cells were passaged twice a week by trypsination (0.25% trypsin, 1 mM EDTA in PBS) and were split 1:10. The medium was changed every second or third day. The cell line was regularly tested with the Mycoplasma PCR Primer Set (Stratagene) and found to be free of Mycoplasma. Tissue culture media, FCS, L-glutamine and penicilin and streptomycin are from Bribco BRL, Gaithersburg, Md., USA.
Transient Expression of COS-1 Cells
On day one COS-1 cells were seeded in 143 cm2 petridish with a density of 2xc3x97104 cells/cm2 in growth medium. At day 2 the cells were co-transfected with 5 xcexcg (total) of experimental plasmid DNA, expressing the FLAG-p38xcex1 and FLAG-MKK6(EE). The plasmids were introduced into the COS-1 cells in serum-free medium using DOTAP(trademark) (Boehringer-Mannheim, Mannheim, Germany). Plasmid DNA was prepared and purified using the QIAGEN EndoToxin-free Maxiprep-500 kit (Hilden, Germany). Briefly, DNA and DOTAP(trademark) were mixed for exactly 15 min. at 37xc2x0 C. in the CO2 incubator. The transfection-mixture was hereafter transferred to a 15-mL falcon-tube and transfection-medium (DMEM with L-Glutamine and Pen./Strep. but without serum) was added to the transfection-mixture, followed by addition to the cell-monolayer. After 4 hours of incubation with DOTAP(trademark) and plasmids, the medium containing double amount of serum was added to the cells bringing the final concentration of serum up to 10%. The cells were then incubated for 24 hours before kinase reaction.
Immunoprecipitation
After 24 hrs of incubation the reaction was stopped by putting the petridish on an ice-bath. The medium was aspirated, and the cell monolayer was washed once in ice-cold PBS (137 mM NaCl, 1.5 mM KH2PO4, 2.7 mM KCl, 8.1 mM Na2HPO4.2H2O), and hereafter solubilised for 10 min. in 1.5 mL lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM Na3VO4, 1% Triton-X-100, Pefabloc 500 xcexcM, Leupeptin 10 xcexcg/xcexcl, Aprotinin 10 xcexcg/xcexcl) was added. The cell-monolayer was scraped by a rubber-policeman, and transferred to an Eppendorf tube. The solubilised cells were clarified by centrifugation at 10.000xc3x97g for 10 min. at 4xc2x0 C. The supernatant was transferred to 50 xcexcl prewashed Protein G Sepharose beads in HNT-buffer (30 mM HEPES, pH 7.5, 30 mM NaCl, 0.1% Triton X-100) and were incubated with 2 xcexcg/sample of monoclonal anti-FLAG(trademark) M2 antibody (raised against the FLAG-epitope, NH2-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-COOH) for 1 hour at room temperature. The anti-FLAG M2 monoclonal antibody was obtained from Sigma (cat. no. F-3165). Approx. 60 xcexcg protein of clarified cell lysate were added to the preadsorbed anti-FLAG(trademark) antibodies on Protein G Sepharose beads and incubated for 90 min. at 4xc2x0 C. in a blood sample mixer. After the immunoprecipitation period the Sepharose beads were washed twice in lysis buffer and twice in a kinase reaction buffer (25 mM HEPES pH 7.5, 10 mM magnesium acetate, 50 xcexcM ATP).
Incubation of the Compounds with Purified p38xcex1 Kinase
The pre-washed immunoprecipitated anti-FLAG-p38 adsorbed on Protein G Sepharose beads was washed twice in 1xc3x97kinase-buffer (25 mM HEPES pH 7.5, 10 mM magnesium acetate, 50 xcexcM ATP), and the supernatant was aspirated. The compounds were diluted in 1xc3x97kinase buffer at the appropriate concentration. The compounds were added to the washed immunoprecipitated and activated FLAG-p38 adsorbed on the Protein G Sepharose beads for 30 min. at 30xc2x0 C. in a volume of 100 xcexcl. Every 10 min. the Eppendorf tubes were tapped to ensure that the beads and the compounds were in the solution. After 30 min. incubation, the beads were spinned down and the supernatant was aspirated.
p38xcex1 MAP Kinase Reaction
The kinase reaction was started by adding 1 xcexcg GST-ATF-2 substrate (Santa Cruz, LaJolla, Calif., USA, cat. no. sc-4114) together with 2 xcexcCi xcex3-32P-ATP in 1xc3x97kinase-buffer per sample. The reaction was allowed to proceed for 30 min. at 30xc2x0 C., and it was stopped by adding 40 xcexcl of 2xc3x97SDS-sample buffer to the kinase reaction. The samples were boiled, spinned down, and resolved on a 15% SDS-PAGE. The dried SDS-PAGE gel was exposed to a Phospho-Imager screen and the radioactive PHAS-1 bands were quantified by the STORM860 Phospho-Imager (Molecular Dynamics, Sunnyvale, Calif., USA) using the ImageQuaNT software.
The inhibition exhibited by compound 114 in this assay is shown in table 2 below:
Reference compound SB 203580 is a widely used reference compound for p38xcex1 MAP kinase inhibition. The compound is commercially available at Calbiochem (Calbiochem-Novabiochem Lajolla, Calif., USA)
These results show, that the compounds of this invention are potent p38xcex1 MAP kinase inhibitors with an improved pharmacological activity compared to a reference compound, thus making them potentially useful in the treatment of inflammatory diseases.
To study the compounds of the present invention in vivo the 12-O-tetradecanoylphorbol-13-acetate (TPA) induced murine chronic skin inflammation model can be used (De Young, L. M. et al., Agents Actions 26, 335-341 (1989); Carlson, R. P. et al., Agents Actions 17, 197-204 (1985); Alford, J. G. et al., Agents Action 37, (1992); Stanley, P. L. et al., Skin Pharmacol. 4, 262-271 (1991)), cf. the description of method in WO 98/32730 hereby incorporated by reference. These results show that compounds of the present invention are of the same potency compared to known reference compounds, e.g. hydrocortisone with its known side effects, whereas the compounds of the present invention are well tolerated and are non-toxic. Some members of the present class of compounds show a very low absorption, thus making them especially useful in the treatment of various dermatological diseases. In general, they may be administered by e.g. oral, intravenous, intranasal, topically or transdermal routes.
The compounds of the present invention may be prepared in a number of ways well known to those skilled in the art of organic synthesis. The compounds of the present invention may be synthesised using the methods outlined below, together with methods known in the art of synthetic organic chemistry, or variations thereof as appreciated by those skilled in the art. Preferred methods include, but are not limited to, those described below.
The novel compounds of formula I, Ia, and Ib may be prepared using the reactions and techniques described in this section. The reactions are performed in solvents appropriate to the reagents and materials employed and are suitable for the transformations being effected. Also, in the synthetic methods described below, it is understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of experiment and work-up procedures, are chosen as standard conditions for that reaction, which should be readily recognised by one skilled in the art. It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the educt molecule must be compatible with the reagents and reactions proposed. Not all compounds of formula I falling into a given class may be compatible with some of the reaction conditions required in some of the methods described. Such restrictions to the substituents which are compatible with the reaction conditions will be readily apparent to one skilled in the art and alternate methods can be used.
The following abbreviations have been used throughout this specification: BINAP=racemic or non-racemic 2,2xe2x80x2-bis(diphenylphosphino)-1,1xe2x80x2-binaphthyl, CDCl3=deuteriochloroform, DMF=N,N-dimethyl-formamide, DMSO-d6=hexadeuterodimethylsulfoxide, DMSO=dimethylsulfoxide, EtOAc=ethyl acetate, Et2O=diethylether, Pd2(dba)3=tris(dibenzylideneacetone)dipalladium(0), mCPBA=meta-chloroperbenzoic acid, MeOH=methanol, NaOt-Bu=sodium-tert-butoxide, KOt-Bu=potassium-tert-butoxide THF=tetrahydrofurane, TLC=thin layer chromatography. 
L: F, Cl, Br, I, or OSO2CF3 
Y: Cl, Br, I, OSO2Rxe2x80x2
FGI: Functional group interconversion
Compounds according to the present invention may be prepared by a process comprising coupling of an amine of the formula III with an bromide, iodide, fluoride, chloride or triflate with the formula II, as shown in Scheme 1, where R1, R2, R3, R4, R5, and X are as defined in general formula I, except that any substituents or functional group which are potentially reactive in the coupling reaction may themselves be protected before the coupling reaction is performed and subsequently removed.
The coupling reaction is carried out using any of the methods for the formation of diphenylamines known to one skilled in the art of organic synthesis.
The preferred method is the palladium catalysed amination method which comprises coupling of an amine with an heteroarylhalogenide (or heteroaryltriflate) in the presence of a base, a suitable Pd source, and a suitable phosphine ligand in an inert solvent. (References: Wolfe, J. P.; Wagaw, S.; Buchwald, S. L.; J. Am. Chem. Soc., (1996), 118, 7215-16; Wagaw, S.; Buchwald, S. L.; J. Org. Chem., (1996), 61, 7240-41; Wolfe, J. P.; Buchwald, S. L.; Tetrahedron Lett., (1997), 38, 6359-62; Hong. Y. et al., Tetrahedron Lett., (1997), 38, 5607-10).
The palladium compound used in the process is not particularly limited, and as specific examples are palladium(II) acetate, palladium(II) chloride, palladium(II) bromide, dichlorobis(triphenylphosphine)palladium(II), tetrakis(triphenylphosphine)palladium(0), tris(dibenzylideneacetone)dipalladium(0). The preferred ligand include, but are not limited to, racemic or non-racemic 2,2xe2x80x2-bis(diphenylphosphino)-1,1xe2x80x2-binaphthyl (hereinafter referred to as BINAP), tri-o-tolylphosphine, tri-tert-butylphosphine, 1,1xe2x80x2-bis(diphenylphosphino)-ferrocene, bis[(2-diphenylphosphino)phenyl]ether (DPEphos), 2-dicyclohexylphosphanyl-2xe2x80x2-dimethylaminobiphenyl, 2-(di-tert-butylphosphino)biphenyl, and 9,9-dimethyl-4,6-bis(diphenylphosphino)xanthene (Xantphos). The amount of palladium and ligand used in this process is typically in the range 0.1 to 10% by mole relative to the amount of the aromatic halide (or triflate) used.
Especially NaOt-Bu and caesium carbonate (Cs2CO3) have proven to be the best bases in this process, but other bases may be used as well.
The reaction is typically performed at elevated temperature (80-120xc2x0 C.) in inert solvents like 1,4-dioxane, toluene, benzene and tetrahydrofurane under an inert atmosphere like argon or nitrogen.
The coupling reaction may also be carried out by nucleophilic substitution of a heteroarylhalogenide with an amine, either in the presence of a base in a polar aprotic solvent, or without a solvent. The preferred base is KOt-Bu or NaH, but other bases may be used as well. The preferred solvent is dimethyl sulfoxide, but other solvents such as DMF may be used as well. The reaction is carried out at elevated temperature (120xc2x0 C.-150xc2x0 C.) for 12-24 h.
Compounds according to the present invention in which R4 is not hydrogen may be prepared by a process comprising coupling of an amine of the formula I (R4=H) with an alkylating agent, as shown in Scheme 1, where R1, R2, R3, R5, and X are as defined in general formula I, except that any substituents or functional group which are potentially reactive in the coupling reaction may themselves be protected before the coupling reaction is performed and subsequently removed.
Typically alkylating agents of the general formula Rxe2x80x94Y include, but are not limited to, iodides (Y=I), bromides (Y=Br), chlorides (Y=Cl) and sulfonates (Y=OSO2Rxe2x80x2, where Rxe2x80x2 represents methyl, trifluoromethyl or 4-methylphenyl).
Compounds according to the present invention may in special cases be prepared by a simple functional group interconversion (FGI), meaning a standard process, known to those skilled in the art of organic synthesis, where a functional group in compounds with the general formula I is transformed into a different functional group in one or more synthetic steps, leading to a new compound with the general formula I. Examples of such processes are, but are not limited to, hydrolysis of an ester to give an acid under basic conditions; deprotection of an methylether to give an phenol by treatment with e.g. borontribromide (BBr3); catalytic hydrogenation of an olefin to give an saturated hydrocarbon and reduction of a nitro group to give an amine.
Compounds according to the present invention with the general formula I were X=S may be prepared from the ketone (with the general formula I, C=O) by such a FGI process, by using one of the many thiocarbonylating reagent, known to those skilled in the art of organic synthesis. Examples of such thiocarbonylating reagents include, but are not limited to, phosphorous pentasulfide (P4S10), or Lawesson""s reagent (2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiaphosphetane-2,4-disulfide) or the like. Compounds accordingly to the present invention with the general formula I were X=Nxe2x80x94OH may be prepared from the ketone (with the general formula I, C=O) by treatment with hydroxylamine, or a protected derivative thereof followed by deprotection, in an appropriate solvent like e.g. pyridine or methanol. 
Compounds according to the present invention with the general formula III may be prepared by several methods known to those skilled in the art of organic synthesis. One useful sequence is shown in Scheme 2. The key step comprising coupling of a bromide (or iodide) with the general formula VI with an acid chloride with the general formula V to afford the benzophenone with the general formula IV. This compound IV may then be reduced to the corresponding amine with the general formula III by treatment with standard reducing agents. Examples of such reducing agents include, but are not limited to, stannous chloride dihydrate; hydrogen, ammonium formiate, or hydrazine hydrate and a catalytic amount of palladium on carbon. The coupling reaction is done by transforming the bromide (VI) into a reactive organometallic intermediate, e.g. by treatment with butyllithium to afford the lithium derivative or by treatment with magnesium to afford the magnesium derivative. The reactivity of this intermediate is then modulated by transmetalation to e.g. zinc, by treatment with ZnCl2, ZnBr2, or ZnI2. This organozinc compound is then coupled with the acid chloride, with the general formula V, under the influence of a palladium 0) complex in catalytic amount. Examples of such catalyst include but are not particularly limited to tetrakis(triphenylphosphine)palladium(0), tetrakis(triphenylarsine)palladium(0), dichlorobis(triphenylphosphine)palladium(II), or benzylchlorobis(triphenylphosphine)palladium(II).
As shown in Scheme 2 compounds with the general formula IV (X=O) may be transformed by a FGI process to give compounds with the general formula IV (X=S or X=Nxe2x80x94OH) as described above. This is only to illustrate the flexibility in the synthesis and in general the described sequence of processes is only one of many possible strategies for the synthesis of compound of the present invention. That is, it may be more advantageous in some cases to alter the sequence of the processes described above. The described sequence of processes is not considered as being limited for the preparation of the compounds of the present invention with the general formula I and alteration of the reaction sequence is an obvious alternative for those skilled in the art of organic synthesis.
In another aspect, the invention relates to a pharmaceutical composition comprising, as an active component, a compound of formula I, Ia, Ib or Ic together with a pharmaceutically acceptable excipient or carrier. The term xe2x80x9cpharmaceutically acceptablexe2x80x9d is intended to indicate that the excipent or carrier included in the composition is compatible with the other ingredients and not toxic or otherwise deleterious to a patient to whom the composition is administered.
Pharmaceutical compositions of the invention may be in unit dosage form such as tablets, pills, capsules, powders, granules, elixirs, syrups, emulsions, ampoules, suppositories or parenteral solutions or suspensions; for oral, parenteral, opthalmic, transdermal, intra-articular, topical, pulmonal, nasal, buccal or rectal administration or in any other manner appropriate for the formulation of anti-inflammatory compounds and in accordance with accepted practices such as those disclosed in Remington: The Science and Practice of Pharmacy. 19th Ed., Mack Publishing Company, 1995. The term xe2x80x9cunit dosagexe2x80x9d is intended to indicate a unitary, i.e. a single dose which is capable of being administered to a patient, and which may be readily handled and packed, remaining as a physically and chemically stable unit dose comprising either the active component as such or a mixture of it with solid or liquid pharmaceutical excipients or carriers. In the composition of the invention, the active component may be present in an amount of from about 0.1-100% by weight of the composition.
For oral administration in the form of a tablet or capsule, a compound of formula I may suitably be combined with an oral, non-toxic, pharmaceutically acceptable carrier such as ethanol, glycerol, water or the like. Furthermore, suitable binders, lubricants, disintegrating agents, flavouring agents and colourants may be added to the mixture, as appropriate. Suitable binders include, e.g., lactose, glucose, starch, gelatin, acacia gum, tragacanth gum, sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes or the like. Lubricants include, e.g., sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride or the like. Disintegrating agents include, e.g., starch, methyl cellulose, agar, bentonite, xanthan gum or the like.
For the preparation of solid compositions such as tablets, the active compound of formula I is mixed with one or more excipients, such as the ones described above, and other pharmaceutical diluents such as water to make a solid preformulation composition containing a homogenous mixture of a compound of formula I. The term xe2x80x9chomogenousxe2x80x9d is understood to mean that the compound of formula I is dispersed evenly throughout the composition so that the composition may readily be subdivided into equally effective unit dosage forms such as tablets or capsules. The preformulation composition may then be subdivided into unit dosage forms containing from about 0.05 to about 1000 mg, in particular from about 0.1 to about 500 mg, of the active compound of the invention.
Liquid formulations for either oral or parenteral administration of the compound of the invention include, e.g., aqueous solutions, syrups, aqueous or oil suspensions and emulsion with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil. Suitable dispersing or suspending agents for aqueous suspensions include synthetic or natural gums such as tragacanth, alginate, acacia, dextran, sodium carboxymethylcellulose, gelatin, methylcellulose or polyvinylpyrolidone.
For parenteral administration, e.g. intramuscular, intraperitoneal, subcutaneous or intravenous injection or infusion, the pharmaceutical composition preferably comprises a compound of formula I dissolved or solubilised in an appropriate, pharmaceutically acceptable solvent. For parenteral administration, the composition of the invention may include a sterile aqueous or non-aqueous solvent, in particular water, isotonic saline, isotonic glucose solution, buffer solution or other solvent conventionally used for parenteral administration of therapeutically active substances, in particular antiproliferative agents. The composition may be sterilised by, for instance, filtration through a bacteria-retaining filter, addition of a sterilising agent to the composition, irradiation of the composition, or heating the composition. Alternatively, the compound of the invention may be provided as a sterile, solid preparation, e.g. a freeze-dried powder, which is dissolved in sterile solvent immediately prior to use.
The composition intended for parenteral administration may additionally comprise conventional additives such as stabilisers, buffers or preservatives, e.g. antioxidants such as methylhydroxybenzoate or the like.
Compositions for rectal administration may be in the form of a suppository incorporating the active ingredient and a carrier such as cocoa butter, or in the form of an enema.
Compositions suitable for intra-articular administration may be in the form of a sterile aqueous preparation of the active ingredient which may be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems may also be used to present the active ingredient for both intra-articular and ophthalmic administration.
Compositions suitable for topical administration, including eye treatment, include liquid or semi-liquid preparations such as liniments, lotions, gels, applicants, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes; or solutions or suspensions such as drops. For topical administration, the active component preferably comprises from 1% to 20% by weight of the composition, but the active ingredient may comprise as much as 50% w/w.
Compositions suitable for administration to the nasal or buccal cavity or for inhalation include powder, self-propelling and spray formulations, such as aerosols and atomizers. Compositions suitable for nasal or buccal administration may comprise 0.1% to 20% w/w. for example about 2% w/w of active ingredient.
The composition may additionally comprise one or more other active components conventionally used in the treatment of various inflammatory diseases and conditions. Examples of such additional active components may be selected from the group consisting of glucocorticoids, vitamin D and vitamin D analogues, antihistamines, platelet activating factor (PAF) antagonists, anticholinergic agents, methylxanthines, xcex2-adrenergic agents, COX-2 inhibitors, salicylates, infomethacin, flufenamate, naproxen, timegadine, gold salts, penicillamine, serum cholesterol lowering agents, retinoids, zinc salts and salicylazosulfapyridine.
In a further aspect, the invention relates to the use of a compound of formula I for the preparation of a medicament for the treatment or prophylaxis of inflammatory diseases or conditions. In a still further aspect, the invention relates to a method of treating inflammatory diseases or conditions, the method comprising administering, to a patient in need thereof, an effective amount of a compound of formula I.
A suitable dosage of the compound of the invention will depend, inter alia, on the particular compound selected for the treatment, the route of administration, the age and condition of the patient, the severity of the disease to be treated and other factors well known to the practising physician. The compound may be administered either orally or parenterally according to different dosing schedules, e.g. daily or with weekly intervals. In general a single dose will be in the range from 0.01 to 400 mg/kg body weight, such as from 0.1 to 100 mg/kg body weight. The compound may be administered as a bolus (i.e. the entire daily dosis is administered at once) or in divided doses two or more times a day.
Inflammatory diseases or conditions contemplated for treatment with the present compounds are inflammatory diseases where modulation of cytokine expression and secretion may be mediated by MAP kinases such as the p38 MAP kinase as discussed above. Examples of inflammatory diseases or conditions believed to be mediated by the p38 MAP kinase are selected from the group consisting of asthma, allergy, arthritis, including rheumatoid arthritis, osteoarthritis and spondyloarthritis, gout, atherosclerosis, inflammatory bowel diease, Crohn""s disease, proliferative and inflammatory skin disorders, such as psoriasis, atopic dermatitis and acne vulgaris, uveitis, sepsis, septic shock, AIDS related diseases and osteoporosis.
The treatment may additionally involve administration of one or more other anti-inflammatory active components such as glucocorticoids, vitamin D and vitamin D analogues, antihistamines, platelet activating factor (PAF) antagonists, anticholinergic agents, methylxanthines, xcex2-adrenergic agents, COX-2 inhibitors, salicylates, infomethacin, flufenamate, naproxen, timegadine, gold salts, penicillamine, serum cholesterol lowering agents, retinoids, zinc salts and salicylazosulfapyridine.